Methods for the controlled expression of DNA fragments in lactic acid bacteria have already been described. However, these known Systems are not satisfactory for various reasons.
For instance, J. M. Wells et al., Appl. Environin. Microbial. 59 (1993) 3954-3959 describe a system for regulation of gene expression by an extracellular factor, in which system the heterologous T7 RNA polymerase gene is brought to controlled expression under the control of the lac promoter from the lactose operon of L. lactis. With this procedure the expression in induced by replacing glucose by lactose in the culture medium. However, this system--and comparable systems from the prior art in which the gene expression is induced by changing the sugar source in the culture medium--has a number of disadvantages, in particular in the case of large-scale preparation of, for example, foodstuffs, such as:
The achievable degree of overexpression is restricted, usually to a factor of at most a few tens. PA1 The regulation is not strict/absolute, that is to say without the presence of the inducer some transcription of the regulated DNA fragment still takes place. PA1 The systems require the use of specific sugar sources, which can be expensive, such as, for example, xylose, which, moreover, have to be added in preponderant amounts. PA1 The sugar inducer can be added effectively only after removal of the sugar used beforehand. PA1 The presence of specific sugars in the end product is not always desirable, whilst the removal thereof--if this is even possible--is associated with additional production steps and additional costs. PA1 In some cases heterologous DNA must be introduced; for instance, the T7 RNA polymerase gene and promoter are used in the abovementioned system. PA1 "This program of starter improvement would be enhanced by the development of procedures enabling controlled expression of specific genes. To achieve this a detailed knowledge of the regulation of gene expression in these bacteria Lactococci! is required. . . . .! However to our knowledge there are no published reports of detailed study on the regulation of gene expression in Lactococci." PA1 Moreover, it is stated on page 6, lines 20-27, that "the Lactobacillus coat protein is apparently synthesized under a wide variety of culture conditions . . . .!. The use of this promoter provides an advantage when it is desired to increase the expressions of genes or operon of, for example, biosynthetic or degradative pathways which are otherwise tightly regulated". PA1 "while the lactose operon is subjected to negative regulation it is only partially switched off during repression. more effective genetic switches are desirable . . .!". PA1 the DNA fragpent is under the control of a promoter of a microbial gene which codes for an antimicrobial paptide, and PA1 the genes coded for on the DNA fragment are brought to expression by the addition of a suitable inducer for the transcription activation. PA1 "While the lactose operon is subject to negative regulation it is only partially switched off during repression. More effective genetic switches are desirable . . .!". PA1 have been brought under the control of a promoter of a microbial gene (cluster) which codes for an antimicrobial peptide, and which PA1 differ from the genes which are naturally--i.e. in the native form or the wild type--regulated by this promoter.
Lakehmidevi et al., Applied Environmental Microbiology, Apr. 1990, p. 934-942 confirm the problems which prior to the present application existed in this specialist field:
Lakshmidevi himself describes the promoter of the bacteriophage Bk5-T which is effactive in Lactococcus. However, this is not a bacterial promoter, nor a promoter which controls the expression of an antimicrobial peptide. Moreover, the bacteriophage Bk5-T DNA is heterologous with respect to lactic acid bacteria and it is also not possible to obtain a strict/absolute regulation of the expression.
In H. Israeleen et al.: "Environmentally regulated promoters in lactococci", Abstract 16, 4th Conference on Streptococcal genetics, Santa Fe, May 15-18 (1994) various induction systems are described for L. lactis, the gene expression being controlled by changing the temperature, growth phase, oxygen concentration and/or substrate components.
A temperature-sensitive regulation of the expression of the dnaJ gene of L. lactis has also been described (J. Bacteriol. 175 1993! 1637-1644).
International Patent Application 94/00581 (Viagen Oy) describes a Lactobacillus expression system based on the gene sequences of the surface protein (sp) gene.
However, this is not a gene that codes for an antimicrobial peptide. Furthermore, the relevant promoter or the relevant operon is not induced by its own gene product.
The gene expression system described in said application therefore does not give a very stringent regulation, whilst the aim of the present invention is to obtain a very strict/absolute regulation.
D. Kirsch et al., Journal of Antibiotics, Part 2, No. 91, 1991, describe an assay for the detection of antibacterial antibiotics, wherein said antibiotics are added to micro-organisms which contain fusions between promoters which can be initiated by antibiotics and structural genes for E. coli .beta.-galaotosidase.
The promoters mentioned in this context are the tetA/tetR promoter of transposon Tn10 induced by tetracycline, the promoter of S. aureus erm C. erythromycin-resistance gene induced by erythromycin and the promoter of S. aureus cat 86 chloramphenicol resistance gene induced by chloramphenicol.
However, these are promoters of resistance genes; such promoters can, of course, be induced by the addition of the relevant antibiotic. However, these antibiotics are not antimicrobial peptides, nor are they the gene product of the relevant resistance genes, nor are they food-grade.
International Patent Application 87/00203 (Columbia University) describes plasmids which contain promoters for bacteriocins such as colicin. These promoters can be used to regulate the expression in E. coli. However, regulation of the expression in lactic acid bacteria using this promoter is neither described nor suggested.
Also, these promoters cannot be induced by their own gene product, that is to say colicin itself and/or derivatives thereof. According to said application, the colicin synthesis can also be induced by a large number of different factors, such as factors in the culture medium, changes in the temperature, and, for example, mitomycin C. There is therefore no question of a strict/absolute regulation.
International Patent Application 92/04451 (Genesit Oy) describes, in general terms, vectors for promoters which are effective in E. coli, B. subtilis, Lactococci and Lactobacillus, chosen from the plasmids pKHT1734 and pKHT1736 described therein.
Neither the nisA promoter, nor any other Lactococcus promoter, the induction thereof or the use thereof for Lactococci is either described or suggested in said application.
However, with these systems the degree of trancription activation is limited and the regulation is not strict, as a result of which the usability for industrial applications is limited.
Furthermore, the addition of unconventional substrate components--such as cooking salt--is undesirable in production processes in the food industry, whilst temperature induction in lactic acid bacteria with, for example, temperature-sensitive repressor systems--for sample as described for E. coli--is not always possible because lactic acid bacteria have a limited temperature range.
Other known systems for regulating the expression in lactic acid bacteria require inducers which are toxic and therefore not acceptable for foodstuffs, such as mitomycin C.
The systems described above also have the disadvantage that with these systems the regulation is generally negative regulation, that is to say repression of the gene expression during growth under specific conditions, which is less simple to use (more laborious) than the use of positive regulation by adding a specific substance at a desired point in time.
Furthermore, in cases where lactic acid bacteria are used in foodstuffs, such as, for example, in cheese and yoghurt, it is undesirable first to make substantial changes to the composition of the medium in order to make it is possible to regulate the transcription of a specific gene, and this is a disadvantage of all of the systems described above.
There is therefore a need for a simple method for the controlled expression of a DNA fragment coding for one or more genes in lactic acid bacteria, with which method the above-mentioned disadvantages can be overcome, so that the transcription of the one or more desired genes can be regulated positively and to a marked degree at any desired point in the growth of the lactic acid bacteria concerned, without adversely influencing the composition of the medium and thus also the desired product characteristics of the fermented product to be obtained.
This certainly also applies in cases where lactic acid bacteria are used as production organisms for foodstuffs or constituents thereof where the production has to take place in a controlled manner.
It has now been discovered that a system of this type can be obtained by making use of promoters of microbial genes which code for antimicrobial peptides from lactic acid bacteria, such as the promoter of the nisin A structural gene.
This structural gene, as well as its promoter and the sequence thereof, have already been described in the prior art Buchman et al.; J. Biol. chem. 263 (31) (1988) 16260-16266!.
International Patent Application 93/20213 (Agriculture and Food Research Council) also describes the sequence of the nisA promoter and its flanking regions, as well as plasmids which contain this promoter.
However, the said application relates to organisms and methods for the production of variant nisins, including nisin Z, which in general are produced by mutating and/or completely inactivating the nisA structural gene.
However, the expression of these mutated nisin structural genes is preferably carried out under the influence of a lacA promoter; to this and the naturally occurring nisin promoter is replaced by said heterologous promoter.
However, the problem addressed by the present application, that is to say the provision of a preferably homologous expression system in Lactococcus, which is able to give a strict/absolute regulation of the gene expression using (in low concentrations) suitable inducers, which preferably are acceptable for food products, is not solved by the said application.
This is also apparent from a discussion of International Patent Application 93/20213 by one of its inventors, M. J. Gasson, in FEMS Microbiol. Rev. 12 (1993), 3-20, in which it is stated that
J. R. van der Meer, O. P. Kuipers, W. M. de Vos et al., Journal of Bacteriology, May 1993, p. 2578-2588 describe the nisP and nisR genes of L. lactis. In this article it is stated that the nisR gone product is a "protein regulatory involved in nisin biosynthesis".
However, the nisR gene product is not a protein with antibacterial activity but a factor which--together with the nisK gene product--is involved in the signal transduction of extracellular nisin, which results in transcription activation. Moreover, although the nisR gene does contain a promoter, this promoter in not induced by the nisR gene product, nor by nisin or its derivatives. Moreover, the nisA and nisZ promoters cannot be induced by the nisR gene product.
It has now been discovered that promoters which code for antimicrobial peptides, and in particular the nisA promoter and the nisZ promoter, can be induced by their own gene product, that is to say the antimicrobial peptide for which they regulate the expression in nature, and/or derivatives thereof. Thus, it has ben found that the nisA promoter can be induced Strictly/absolutely by nisin or suitable analogues/derivatives thereof, for example derivatives obtained after post-translational expression, By this means it is possible, by adding such an inducer/inducing factor in a controlled manner at a desired point in time, to obtain expression of a homologous or heterologous gene which is operably connected to said antimicrobial promoters.